Human tumors are immunogenic and many tumor antigens have been identified. A problem now facing tumor immunologists is to determine what antigens are relevant to the destruction of tumor tissue, i.e. tumor rejection antigens. Ideal tumor rejection antigens would be ones that [1] are essential to the underlying biology of the tumor, [2] are the targets of a multifaceted immune response of high magnitude consisting of T helper cells, cytolytic T cells, and antibodies, and, [3] those to which an immune response confers therapeutic benefit. Previous studies from our laboratory have shown that stage IV breast cancer patients can be successfully immunized with HER-2/neu (HER2) peptide vaccines resulting in epitope spreading and improved survival. Both intramolecular and intermolecular epitope spreading occurred as a result of immunization. In autoimmune disease, epitope spreading is associated with aggressive tissue specific destruction. Epitope spreading may explain why our patients had improved survival. Identifying those antigens to which epitope spreading occurred in this population of stage IV breast cancer patients may prove useful in developing cancer vaccine strategies to target and specifically destroy tumor tissue. Our hypothesis is that these antigens may be tumor rejection antigens. The specific aims of this proposal are to: (1) identify potential "tumor rejection" antigens using sera derived from breast cancer patients immunized with a HER2 peptide-based vaccine who demonstrated epitope spreading and prolonged survival after active immunization, and, (2) determine whether antigens, identified through serologic screening, can be recognized by T cells derived from breast cancer patients. We will use the technique of serological analysis of recombinant cDNA expression libraries or "SEREX" to screen for tumor-specific antigens that were the result of epitope spreading following immunization. Many of the tumor specific IgG antibody responses identified in this fashion are high titer implying recognition by CD4+ T helper cells. Thus, it is assumed that antigens discovered using antibody immunity as a screening tool can be considered to be recognized by the CD4+ T helper cell repertoire (2) and, potentially, CTL. We will use post-immunization sera derived from Stage IV breast cancer patients that had both improved survival and epitope spreading following immunization with a HER2 peptide vaccine to identify novel immunogenic proteins. New antigens induced by immunization will be identified by "subtraction" of antigens recognized by the pre-vaccine serum. Subsequent studies will determine whether these new antigens, identified via serologic screening, can serve as T cell targets. Our goal is to develop immune-based strategies (e.g. vaccines) that would be associated with tumor destruction and survival benefit.